Effects of Teucrium Polium Aerial Parts extract on oral glucose tolerance tests and pancreas histopathology in Streptozocin-induced diabetic rats.

Teucrium polium can reduce serum glucose. There are few reports in the literature related to this subject and the resolution of this mechanism requires further experiments. The aim of the present study was to evaluate the effects of Teucrium polium aerial parts extracts on oral glucose tolerance tests and pancreas histology in streptozocin-induced diabetic rats. In order to prepare the aqueous concentrate, aerial parts extract was dissolved in distilled water and was boiled for 30 minutes. For the preparation of ethanolic solution, powder was dissolved in ethanol and mixed by a shaker. Diabetic rats were induced with single IP injection of streptozotocin (STZ) at a dose of 50 mg/kg body weight dissolved in normal saline just before use to the 16 hr fast rats. Both groups, diabetic and normal were sacrificed by ether anesthesia. The tissue samples were formalin fixed and paraffin embedded for microscopic examination in accordance with routine laboratory procedures. Blood was collected from the tail vein of the rats. Serum glucose levels were then measured by commercial kits by using a glucose oxidized method. There were no biochemical abnormalities or histological changes in the pancreas of control rats. Post treatment of Teucrium polium aerial parts extract reduced the severity of streptozotocin diabetic pancreases. Our histopathological investigation along with the biochemical evaluations showed a significant effect on histological changes in the pancreas of induced diabetic rats upon Teucrium polium aerial parts extract treatment (P<0.05).

It was previously shown that Teucrium polium crude extract chemical-induced diabetic rats can significantly decrease serum glucose values (1,2). It has also been demonstrated that administration of a hydro-alcoholic extract of Teucrium polium by gavages enhances serum insulin level in rats (3). As reported by investigators extract of Teucrium polium has hypoglycemic activity in diabetic animals (4). Previous work has indicated that the combinations of Teucrium polium with anticancer drugs may be able to increase capability of cancer chemotherapies. There is accumulating evidence that streptozocin-induced diabetes resulted in a decrease in the number of pancreatic islets (5,6).
In a recent investigation the possibility of the islets regeneration upon plant extract treatment was studied. However, any significant improvement in lipid metabolism was not shown after Teucrium polium extract treatment (7). In literature , it was reported that administration of medical plants might be useful in preventing hyperglycemia in rabbits (8). Some data have revealed that traditional medicine can be hepatotoxic (9). Many investigators have shown that Teucrium polium extract reduces blood glucose via mechanisms such as enhancement of peripheral metabolism of glucose rather than an increase in insulin release (10). Previous study reported that the aqueous extract of Teucrium polium reduced the serum levels of lipids in rats (11). Evidence revealed that methoxylated flavonoids exhibited a lesser antioxidant activity (12).
Based on the mentioned studies, it may therefore be interesting to evaluate the effects of Teucrium polium aerial parts extracts on oral glucose tolerance tests and pancreas histology in streptozocin-induced diabetic rats. The present experiments were undertaken to evaluate the effects of Teucrium polium aerial parts extracts on biochemical and histological parameters.

Plant Materials
In order to prepare the aqueous form, 100 g of dry Teucrium polium aerial parts were dissolved in two litres of distilled water and were boiled

Diabetic induction
The rats were not fed 16 h before injection.
The streptozotocin (STZ) solution was prepared and 0.6ml volume with 50 mg/kg dose was injected intraperitoneally to the rats in the hyperglycemic group. The rats with fasting glycemia more than 250 mg/dl were used as diabetic group. In addition, 0.7 ml normal saline was injected to rats in the control group.

Animal experiments
A group of 6 rats was considered as the normal group that received normal chow and tap water. The other 18 hyperglycemic animals were divided randomly and equally into three groups of six. The first and second group of diabetic rats (diabetic experimental rats) received the aqueous Teucrium polium aerial parts extract twice a day through a gavage tube for a period of 14 days. The third group of diabetic rats (diabetic control rats) received only water and rat chow ad libitum.

Glucose tolerance test
At the end of the gavage feeding of the extract, half of the rats in each of the two groups were fasted for 14 hours and underwent oral glucose tolerance test (GTT), while the other half were immediately sacrificed by decapitation.
Glucose tolerance test in fasted animals was performed on 16 h fasted rats using one gram glucose/kg body weight. Duplicate blood samples from the animal tails were collected. In all groups, blood was collected by tail snipping at 0, 30, 90 and 120 minutes after glucose load. The glucose concentration data were used to compare glucose tolerance in the various groups.

Histopathologic evaluation
The animals in four groups were sacrificed by ether anesthesia. Whole pancreas was dissected.

Statistical analysis.
All tests were carried out in triplicate.
Statistical analyses were done using SPSS version 16.0. The significance of differences between the mean values were determined by analysis of variance (ANOVA), and a P value of less than 0.05 was considered statistically significant.

Results
In the present study, it is clearly demonstrated that the pancreas from control rat shows normal pancreatic histoarchitecture islet.
There were no abnormalities in the pancreas of control rats. Representative pictures of pancreas histology is shown in Fig.1.
The pancreas from the diabetic rats showed degeneration of serous acini and the numbers of islet and islet cells were found to be lower than the control group.
As it is evident from fig 2, the pancreases are irregularly shaped and relatively small.
Representative picture of pancreas histology is shown in Fig.2.
The pancreas from a diabetic rat treated with powder of ethanolic Teucrium polium aerial parts extract shows slight to moderate regeneration. Fig 3. The pancreas from a diabetic rat treated with powder of aqueous Teucrium polium aerial parts  We conclude that Teucrium polium aerial parts extract stimulate pancreas repair and may be clinically beneficial as an agent to restore or maintain pancreas tissue after injury. The results of this paper can encourage clinical studies to evaluate the potential benefit of Teucrium polium aerial parts extract administration.